Tuned synthetic dendrimer calibrants for mass spectrometry

ABSTRACT

Provided are synthetic dendrimer calibrants for mass spectrometry. The calibrants are distinguished by their relative case and rapidity of synthesis, comparatively low cost, long shelf life, high purity, and amenability to batch synthesis as mixtures. The latter characteristic enables parallel preparation of higher molecular weight compounds displaying useful distributions of discrete molecular weights, thereby providing multi-point mass spectrometry calibration standards. Methods of making, tuning and using said calibrants are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 14/402,474,filed Nov. 20, 2014, which is a U.S. national stage entry ofInternational Application No. PCT/US2013/042110, filed May 21, 2013,which claims the priority benefit of U.S. Provisional Application No.61/649,920, filed May 21, 2012, all of which are incorporated herein byreference in their entireties.

BACKGROUND 1. Field

The present disclosure relates to dendritic molecules havingserially-branched structure wherein at least one of the branchespossesses a second branching structure. The present disclosure alsocomprises methods for the preparation of said dendritic molecules, theiruse as calibrants for time-of-flight matrix-assisted laserdesorption/ionization (MALDI-TOF) mass spectrometry (MS), electrosprayionization (ESI-MS), atmospheric pressure chemical ionization (APCI-MS),fast atom bombardment (FAB-MS), and other MS techniques for the analysisof compounds with molecular weights greater than 1000 Daltons. Thepresent disclosure further relates to the tuning of dendritic molecules,the method of preparation of said tuned dendritic molecules, and theiruse as calibrants.

2. Description of Related Art

Mass spectrometry (MS) is an analytical technique for determining theelemental composition of samples (e.g., proteins, chemical compounds,etc.). It may also be used in determining the chemical structures ofsuch samples. Generally, MS comprises ionizing a sample to generatecharged molecules (and fragments thereof), and measuring theirmass-to-charge ratios.

Time-of-flight mass spectrometry (TOF-MS) is a method in which ions areaccelerated by an electric field into a field-free drift region with akinetic energy of qV, where q is the ion charge and V is the appliedvoltage. Since each ion's kinetic energy is ½mv², where m is mass and vis velocity, lighter ions have a higher velocity than heavier ions.Thus, the lighter ions reach the detector at the end of the drift regionsooner than the heavier ions. Matrix-assisted laserdesorption/ionization (MALDI) is an ionization technique used in massspectrometry, which facilitates the analysis of biomolecules (e.g.,proteins, peptides, and sugars) and large organic molecules (e.g.,polymers and other macromolecules).

Electrospray ionization (ESI) is an atmospheric pressure ionizationtechnique whereby an analyte, dissolved in volatile solvent (e.g.,acetonitrile, CH₃OH, CH₃Cl, water, etc.), is forced through a small,charged capillary (usually metal). The analyte exists as an ion insolution, and as the sample is forced out of the capillary itaerosolizes. This increases the distance between the similarly-chargedanalyte particles. A neutral gas carrier (e.g., nitrogen) is often usedto evaporate the solvent from the droplets. As the solvent evaporates,the charged analyte molecules are brought closer together. At the sametime, though, the like charge on the analyte molecules forces themapart. This process of contraction and expansion repeats until thesample is free of solvent and is a lone ion. The lone ion then proceedsto the mass analyzer.

Atmospheric pressure chemical ionization (APCI) is also an atmosphericpressure ionization technique, whereby a sample solution passing througha heated tube (e.g., greater than 400° C.) is volatilized and subjectedto a corona discharge with the aid of nitrogen nebulization. APCI is avariant of ESI, and can be performed in a modified ESI source. Ions,produced by the discharge, are extracted into the mass spectrometer.This technique is best for relatively polar, semi-volatile samples, andmay be used as a liquid chromatography-mass spectrometry (LC/MS)interface because if can accommodate very high liquid flow rates (e.g.,1 mL/min). Spectra from APCI-MS usually contain the quasi-molecular ion[M+H]⁺.

Fast atom bombardment (FAB) employs a high-energy beam of neutral atoms,typically xenon or argon, which strikes a solid sample (analyte mixedwith matrix) under vacuum to cause desorption and ionization. Commonmatrices include glycerol, thioglycerol, 3-nitrobenzyl alcohol (3-NBA),18-Crown6 ether, 2-nitrophenyloctyl ether, sulfolane, diethanolamine,and triethanolamine. FAB is used for large biological molecules that aredifficult to get into the gas phase. The high-energy beam is produced byaccelerating ions from an ion source through a charge-exchange cell.Those ions accumulate an electron through collisions with neutral atoms,to form a beam of high-energy atoms. Because FAB spectra often containonly a few fragments, and a signal for the pseudo molecular ion (e.g.,[M+H]⁺, [M+Na]⁺), it is useful for determining molecular weights. Thelow m/z region, though, is usually crowded with signals from the matrix.

In order to calibrate mass spectrometers for a range of analytical work,including protein, peptide, oligonucleotide, and synthetic polymercharacterization and structural determination, known calibrants of adiverse set of molecular weights are required. Typically, proteins andpeptides have been used because of their monodispersity (only a singleand exact molecular weight is present in a pure sample) and theiravailability from biological sources. Examples include: bradykinin,adrenocorticotropic hormone, insulin chain B, cytochrome c,apomyoglobin, albumin, aldolase, and angiotensin II. However, theproduction—and particularly the purification—of such standards is timeconsuming and technically complicated, leading to a fairly high expensefor gram quantities. In addition, such standards have inherently poorshelf-life due to enzymatic instability and acid sensitivity.

Synthetic polymers offer a much cheaper alternative, but exist as abroad distribution of molecular weights because they are prepared usinga relatively unmediated reaction between single monomer units (comparedto biological syntheses) that inevitably result in a statisticaldistribution of molecular weights. This broad distribution of molecularweights is typically observed in mass spectra as a Gaussian series ofpeaks, evenly spaced as multiples of the monomer mass. However, thedevelopment of efficient dendrimer syntheses offers to marry the cheapscalable cost of synthetic materials with the exact molecular weighttraditionally associated with biosynthesized materials.

Two contrasting synthetic routes towards the preparation of “true”dendrimers (highly branched, molecules with a high degree of structuralregularity) are known.

The first approach—the divergent approach—first involves the coupling ofa branched monomer to a core molecule, yielding an intermediate, andthen “activation” of the intermediate to produce a new, larger moleculewith an enhanced number of surface functionalities. Repetition of thesetwo steps leads to outward, layer-by-layer growth of dendritic moleculeshaving exponentially increasing size.

The second approach the convergent approach involves peripheral groupswhich are tethered via one monomer unit, producing “wedges” or“dendrons.” Two of these dendrons may be coupled with an additionalmonomer molecule to make a larger dendron, and growth continues inward,layer by layer, until coupled to a core.

Typically, divergent techniques are technically simple: a large excessof a small molecule reacts with the growing molecule, and then isremoved (e.g., by distillation), providing a relatively cost-efficientand scalable synthesis. With divergent techniques, however, the numberof coupling reactions increases exponentially with each generation.Consequently, dendrimers with minor structural impurities are nearlyinevitable and cannot be easily removed (e.g., when n is a large number,the product of n coupling reactions has physical properties nearlyidentical to the product of n−1 couplings). The result is poorly-definedmaterials for applications such as MS calibration.

Convergent techniques have the distinct advantage that each couplinginvolves a small and constant number of reactions (usually 2 or 3reactions). Thus, with convergent techniques the reactions can be drivento completion and any impurities generated by side reactions are easilydetected (since n is small) and removed. But while the materialsproduced with convergent techniques are well-defined, their synthesis isdemanding. This prevents their economical use for all but specialtyapplications.

The technical problem underlying the present disclosure was therefore toovercome these prior art difficulties by providing monodispersecalibrants with improved shelf-life, at lower cost, and over a broadrange of molecular weights. The solution to this technical problem isprovided by the embodiments characterized in the claims.

BRIEF SUMMARY

The present disclosure relates to dendritic molecules—dendrimers—usefulfor calibration of mass spectrometry instruments, and particularlyuseful in MALDI-TOF, ESI, APCI, and FAB mass spectrometry techniques andany additional technique used for mass analysis of materials withmolecular weights above 1,000 daltons. The present disclosure alsorelates to methods of synthesizing said dendrimers, as well as methodsof using them.

The disclosure relates, in one aspect, to synthetic calibrants. Thesynthetic calibrants of the present disclosure are dendriticmolecules—dendrimers—synthesized (“generated”) via “dendronization” of ahydroxyl-terminated core molecule and, optionally, a subsequent“deprotection” step. Also optionally, the dendronization anddeprotection steps may be performed multiple times (wherein eachdeprotection step follows a dendronization step, and wherein eachdendronization step after the first dendronization step follows adeprotection step) to yield dendrimers of known and useful sizes. Thedendrimer products of each round of dendronization/deprotection are partof the same “generation.”

For example, the first dendronization step performed with a coremolecule yields a first generation, or “G-1” dendrimer. Likewise, thenext deprotection step performed on the resulting G-1 dendrimer alsoyields a first generation dendrimer. The dendronization step after theG-1 deprotection step, however, leads to a second generation, or “G-2”dendrimer. Thus, each round of dendronization and deprotection yielddendrimer products of the same “generation.” In a preferred embodiment,the disclosure relates to a mixture of dendrimers of different molecularweights, and especially to a specifically proportioned mixture (e.g., anequimolar mixture) of said dendrimers. In particular, the presentdisclosure relates to a mixture of dendrimers synthesized in parallel,wherein equimolar quantities of core molecules bearing different numbersof alcohol functionalities are mixed together and subjected to at leastone round of dendronization. Optionally, the resulting mixture may besubjected to several rounds of dendronization and deprotection to yielddendrimer mixtures of known and useful sizes, across a broad spectrum ofmolecular weights. In each of these mixtures, the dendrimers are of thesame generation and all are useful in mass spectrometry. Additionally,because the end groups can be modified by dendronization anddeprotection, the dendrimers of the present disclosure possess highsolubility in nearly the full spectrum of solvents, matrices, andanalytes useful for MS. Consequently, the dendrimers of the presentdisclosure are useful as internal calibrants (i.e., they may be mixeddirectly with the analyte and matrix during sample preparation).

In one embodiment, a composition is provided comprising a firstdendrimer comprising a first core molecule, wherein said first coremolecule is selected from the group consisting of: a molecule comprisingbetween 1 and 8 alcohol functionalities, a molecule comprising between 1and 8 amine functionalities, and a molecule comprising at least oneamine functionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; a second dendrimercomprising a second core molecule, wherein said second core molecule isselected from the group consisting of: a molecule comprising between 1and 8 alcohol functionalities, a molecule comprising between 1 and 8amine functionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said second coremolecule is at least 2 but no greater than 8; and wherein said firstcore molecule has a different number of total alcohol functionalitiesand amine functionalities than said second core molecule.

In another embodiment, a composition is provided comprising a firstdendrimer comprising a first core molecule, wherein said first coremolecule is selected from the group consisting of: a molecule comprisingbetween 1 and 8 alcohol functionalities, a molecule comprising between 1and 8 amine functionalities, and a molecule comprising at least oneamine functionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; and a second dendrimercomprising a second core molecule, wherein said second core moleculecomprises a subsequent generation dendrimer of said first core molecule.

In yet another embodiment, a method of manufacturing is providedcomprising the steps of: providing a composition comprising a first coremolecule wherein said first core molecule is selected from the groupconsisting of: a molecule comprising between 1 and 8 alcoholfunctionalities, a molecule comprising between 1 and 8 aminefunctionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; a second core moleculewherein said second core molecule is selected from the group consistingof: a molecule comprising between 1 and 8 alcohol functionalities, amolecule comprising between 1 and 8 amine functionalities, a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said second core molecule is at least 2 but nogreater than 8; and wherein said first core molecule has a differentnumber of total alcohol functionalities and amine functionalities thansaid second core molecule; and subjecting said first core molecule andsaid second core molecule to a round of dendronization.

In yet another embodiment a method of manufacturing is providedcomprising the steps of providing a composition comprising a firstdendrimer comprising a first core molecule, wherein said first coremolecule is selected from the group consisting of: a molecule comprisingbetween 1 and 8 alcohol functionalities, a molecule comprising between 1and 8 amine functionalities, and a molecule comprising at least oneamine functionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; and a second dendrimercomprising a second core molecule, wherein said second core moleculecomprises a subsequent generation dendrimer of said first core molecule;and subjecting said first core molecule and said second core molecule toa round of dendronization.

In yet another embodiment a method of determining physical properties ofa sample is provided, the method comprising: providing a compositioncomprising a first dendrimer comprising a first core molecule, whereinsaid first core molecule is selected from the group consisting of: amolecule comprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said first core molecule is at least 2 but no greaterthan 8; a second dendrimer comprising a second core molecule, whereinsaid second core molecule is selected from the group consisting of: amolecule comprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said second core molecule is at least 2 but nogreater than 8; wherein said first core molecule has a different numberof total alcohol functionalities and amine functionalities than saidsecond core molecule; and wherein said composition has physicalproperties; ionizing at least a portion of said composition; providingan analyte sample wherein said analyte sample has physical properties;ionizing at least a portion of said analyte; collecting data from saidionized portion of said composition and said ionized portion of saidanalyte sample; and relating said data to said physical properties ofsaid portion of said composition, thereby determining said physicalproperties of said analyte sample.

In yet another embodiment a method of determining physical properties ofa sample is provided, the method comprising providing a compositioncomprising a first dendrimer comprising a first core molecule, whereinsaid first core molecule is selected from the group consisting of: amolecule comprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said first core molecule is at least 2 but no greaterthan 8; a second dendrimer comprising a second core molecule, whereinsaid second core molecule comprises a subsequent generation dendrimer ofsaid first core molecule; and wherein said composition has physicalproperties; ionizing at least a portion of said composition; providingan analyte sample wherein said analyte sample has physical properties;ionizing at least a portion of said analyte; collecting data from saidionized portion of said composition and said ionized portion of saidanalyte sample; and relating said data to said physical properties ofsaid portion of said composition, thereby determining said physicalproperties of said analyte sample.

In yet another embodiment, a method of calibrating a mass spectrometeris provided, the method comprising providing a composition comprising afirst dendrimer comprising a first core molecule, wherein said firstcore molecule is selected from the group consisting of: a moleculecomprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said first core molecule is at least 2 but no greaterthan 8; a second dendrimer comprising a second core molecule, whereinsaid second core molecule is selected from the group consisting of: amolecule comprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said second core molecule is at least 2 but nogreater than 8; wherein said first core molecule has a different numberof total alcohol functionalities and amine functionalities than saidsecond core molecule; and wherein said composition has physicalproperties; ionizing at least a portion of said composition; collectingdata from said ionized portion of said composition; and relating saiddata to said physical properties.

In yet another embodiment, a method of calibrating a mass spectrometeris provided, the method comprising: providing a composition comprising afirst dendrimer comprising a first core molecule, wherein said firstcore molecule is selected from the group consisting of: a moleculecomprising between 1 and 8 alcohol functionalities, a moleculecomprising between 1 and 8 amine functionalities, and a moleculecomprising at least one amine functionality and at least one alcoholfunctionality wherein the combined number of amine and alcoholfunctionalities of said first core molecule is at least 2 but no greaterthan 8; a second dendrimer comprising a second core molecule, whereinsaid second core molecule comprises a subsequent generation dendrimer ofsaid first core molecule; and wherein said composition has physicalproperties; ionizing at least a portion of said composition; collectingdata from said ionized portion of said composition; and relating saiddata to said physical properties.

BRIEF DESCRIPTION OF THE DRAWINGS

For a further understanding of the nature, objects, and advantages ofthe present disclosure, reference should be had to the followingdetailed description, read in conjunction with the following drawings,wherein like reference numerals denote like elements.

FIG. 1 is a schematic diagram showing the synthesis of tri-functional“C-3” calibrants of the present disclosure.

FIG. 2 is a schematic diagram showing the synthesis of tetra-functional“C-4” calibrants of the present disclosure.

FIG. 3 is a schematic diagram showing the synthesis of penta-functional“C-5” calibrants of the present disclosure.

FIG. 4 is a schematic diagram showing the synthesis of hexa-functional“C-6” calibrants of the present disclosure.

FIG. 5 is a schematic diagram showing the parallel synthesis of tri-,tetra-, penta- and hexa-functional calibrants of the present disclosure.

FIG. 6 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 1, 11, 21, and 31 of the present disclosure.

FIG. 7 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 3, 13, 23, and 33 of the present disclosure.

FIG. 8 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 4, 14, 24, and 34 of the present disclosure.

FIG. 9 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 5, 15, 25, and 35 of the present disclosure.

FIG. 10 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 6, 16, 26, and 36 of the present disclosure.

FIG. 11 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 7, 17, 27, and 37 of the present disclosure.

FIG. 12 shows the results of MALDI-TOF analysis of an equimolar mixtureof dendrimers 8, 18, 28, and 38 of the present disclosure.

FIG. 13A shows the spectrum results of MALDI-TOF analysis of adendronized cavitand (Cav-([G1]-Ph)₈, having molecular formulaC₁₉₂H₁₇₆O₄₈.

FIG. 13B shows the structure of the dendronized cavitand from FIG. 13A.

FIG. 14 shows the results of MALDI-TOF analysis of the PEG 1970 33-mer,having the molecular formula C₆₆H₁₃₄O₃₄.

FIG. 15 shows the results of MALDI-TOF analysis of the PEG 1970 43-mer,having the molecular formula C₈₆H₁₇₄O₄₄.

FIG. 16 shows the results of MALDI-TOF analysis of the PEG 1970 53-mer,having the molecular formula C₁₀₆H₂₁₄O₅₄.

FIG. 17 shows the results of MALDI-TOF analysis of the proprietarypeptide JF-1485, having the formula C₈₈H₁₁₈N₁₆O₂₂S₅₊.

FIG. 18 shows an ESI-mass spectrum of G1 mixture of dendrimers 1(C3-([G-1]Ph)₃), 11 (C4-([G-1]Ph)₄), 21 (C5-([G-1]Ph)₅), and 31(C6-([G-1]Ph)₆). Samples were prepared by dissolving in acetonitrile andinjecting directly without addition of counterion. Residual sodiumyielded the observed mass spectra with a single sodium cation.

FIG. 19 shows an ESI-mass spectrum of G1 mixture of dendrimers 3(C3-([G-2]Ph₂)₃), 13 (C4-([G2]Ph₂)₄), 23 (C5-([G-2]Ph₂)₅), and 33(C6-([G-2]Ph₂)₆). Samples were prepared by dissolving in acetonitrileand injecting directly without addition of counterion. Residual sodiumyielded the observed mass spectra with a single sodium cation fordendrimer 3, as well as doubly-charged complexes (two sodium cations)for dendrimers 13, 23, and 33.

FIG. 20 shows a first view of a peptide population map wherein thegraphical data represents the total population of all possible peptidesper 0.01 u of mass defect for each nominal molecular weight.

FIG. 21 shows a second view of said peptide population map wherein thegraphical data represents the total population of all possible peptidesper 0.01 u of mass defect for each nominal molecular weight.

FIG. 22 shows a third view of said peptide population map wherein thegraphical data represents the total population of all possible peptidesper 0.01 u of mass defect for each nominal molecular weight.

FIG. 23 shows a peptide population map configured to show the scarcineridge wherein the graphical data represents the total population of allpossible peptides per 0.01 u of mass defect for each nominal molecularweight.

FIG. 24 is a schematic diagram showing the synthesis of mono-functional“C-1” iodo-core calibrants of the present disclosure.

FIG. 25 shows the results of MALDI-TOF analysis of iodo-core dendrimer 2of the present disclosure.

FIG. 26 shows the results of MALDI-TOF analysis of iodo-core dendrimer 4of the present disclosure.

FIG. 27 shows the results of MALDI-TOF analysis of iodo-core dendrimer 6of the present disclosure.

FIG. 28 shows the results of an internal calibration test of iodo-coredendrimer 2 against representative peptide Endomorphin T.

FIG. 29 is a schematic diagram showing the synthesis of tri-functional“C-3” amine-core calibrants of the present disclosure.

FIG. 30 shows the results of a MALDI-TOF analysis of amine coredendrimer 2 of the present disclosure.

FIG. 31 shows the results of a MALDI-TOF analysis of amine coredendrimer 4 of the present disclosure.

FIG. 32 is a schematic diagram showing the dendronization ofN,N,N′,N′-Tetrakis(2-hydroxyethyl)ethylenediamine to create C4-[G1]-Ph₄.

FIG. 33 shows the results of a MALDI-TOF analysis of amine coredendrimer C4-[G1]-Ph₄ of FIG. 32.

FIG. 34 is a schematic diagram showing the dendronization of bis-tris tocreate C5-[G1]-Ph₅.

FIG. 35 shows the results of a MALDI-TOF analysis of amine coredendrimer C5-[G1]-Ph₅ of FIG. 34.

DETAILED DESCRIPTION

Before the subject disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments of the disclosure described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the presentdisclosure will be established by the appended claims.

Furthermore, this application incorporates by reference, in theirentireties, U.S. Non-Provisional application Ser. No. 11/290,998, whichis the National Stage of International Application No. PCT/US10/23087filed on 3 Feb. 2012, U.S. Provisional Patent Application No.61/149,506, filed 3 Feb. 2009, U.S. Provisional Patent Application No.61/167,708, filed on 8 Apr. 2009, and U.S. Provisional PatentApplication No. 61/185,665, filed on 10 Jun. 2009.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this disclosurebelongs.

As used herein, the term “[M+Ag]+” indicates that one silver cation isattached per molecule, during ionization of samples, as the counterion.Other counterions may include, for example and without limitation, “H”,“Na”, and “K”, as will be readily appreciated by those persons havingordinary skill in the relevant art. As used herein, the term “m/z”denotes the mass-to-charge ratio. As used herein, “MW” means molecularweight.

The recently developed divergent aliphatic poly(ester) synthesis appearsto offer the advantages of both techniques, while minimizing theshortcomings of both. A divergent dendritic synthesis is an iterativeprocess that involves a well-defined (though exponential) increase ofmass with each repetition of two synthetic steps: the “coupling step,”and the “deprotection step.” In FIG. 1, for example, the “coupling step”(e.g., step “i” in FIG. 1) involves reaction of a specific number ofalcohol functionalities (—OH groups) from the core structure with thebenzylidene protected bis-MPA acid anhydride (IUPAC namebis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydride monomer(“monomer” in FIG. 1) In doing so, an exact number of monomer units areconnected to the core molecules, yielding a new dendritic molecule witha discrete molecular weight. In the “deprotection step” (e.g., step “ii”of FIG. 1), a palladium catalyst (palladium(II) hydroxide supported ongraphite, also known as Pearlman's catalyst) is used to remove thebenzylidene protecting groups via a hydrogenolysis reaction to generatea new core. It should be noted that in doing so, the number of alcoholfunctionalities doubles after carrying out each iteration of couplingand deprotection, thus enabling the process to be repeated and thestructures to grow exponentially but in a well controlled fashion and soyielding monodisperse products. Because the coupling step involves theclean, highly activated esterification reaction of alcohol functionalgroups with acid anhydrides, the reaction can be carried out in“quantitative” yields (greater than 99.9%), without byproduct. Inaddition, a number of deprotection steps (e.g. palladium (“Pd”)catalyzed hydrogenolysis and acid catalyzed hydrolysis for thecorresponding benzylidene and acetal protected monomer) can be carriedout in an equally clean and quantitative fashion, providing monodispersecompounds sufficiently pure to act as calibrants for mass spectrometry.At the same time, this divergent approach offers a fast route that istechnically simple without chromatographic purification, enablingcost-efficient, scalable production.

The synthetic dendrimer calibrants of the present disclosure offer anumber of distinct advantages over other calibrants. Peptides andproteins have been used as commercial standards for calibration because,traditionally, these were the only monodisperse polymers which could beprepared and purified with sufficiently high molecular weight. Whilepeptide and protein calibrants provide a viable standard, they sufferfrom short shelf-life (because of the prevalence of peptidases) and highcost (because their synthesis and purification is typically carried outon a milligram scale). A representative example of these calibrants isprovided in TABLE 1.

TABLE 1 Prior Art Peptide and Protein Calibrants Molecu Price perCalibrant Weight (USD) Bradykinin Fragment 756 38,300 Angiotensin II1,046 6,580 P₁₄R 1,533 8,733,300 ACTH Fragment 18- 2,464 220,500 InsulinChain B 3,496 8,160 Insulin 5,730 2,652,900 Cytochrome c 12,3621,181,000 Apomyoglobin 16,952 861,200 Aldolase 39,211 372,300 Albumin66,429 219,800

Source: Sigma-Aldrich, Inc.

Synthetic calibrants offer a number of potential advantages, includingincreased shelf-life, but until recently the only products that could beproduced at a competitive price were polydisperse polymers (i.e., theyexhibit a broad range of mass characteristics). The presence of multiplespecies (and the prevalence of different counterions in MS, includingMALDI-TOF, ESI, APCI, and FAB) has prevented these from becoming anattractive alternative to peptides and proteins. Monodisperse syntheticcalibrants, such as P₁₄R, are at least 3 times as expensive as thenext-cheapest peptide calibrant (Insulin), and more than 1,000 timesmore expensive than the cheapest peptide calibrant (Insulin Chain B).

The synthetic dendrimer calibrants of the present disclosure, incontrast, are less expensive to produce. Because of this rapid syntheticaccess to cost-efficient, yet highly pure dendritic compounds, thedendrimer calibrants of the present disclosure offer a competitivesolution to the calibration of mass spectrometers, particularly whenusing MALDI-TOF, ESI, APCI, or FAB methods. In addition, they can besynthesized as mixtures, thus reducing preparation, purification, andpackaging costs. While presently-available peptide and proteincalibrants are widely used and accepted, the reduced cost of thedendrimer calibrants of the present disclosure, as well as theirimproved shelf-life and solvent compatibility, should result in theirready acceptance.

The dendrimers are given a standard nomenclature to denote theirarchitecture. For example, in the names “CX-([G-n]Ph_(p))_(z),” and“CX-([G-n]OH_(q))_(z),” the “CX” term refers to the number of alcoholfunctionalities on the core—the “core number”—where “X” is an integer.Thus, “C3” refers to 1,1,1-trishydroxyethylmethane (a triol) as thecore, “C4” refers to pentaerythritol (a tetraol) as the core, “C5”refers to xylitol (a pentaol) as the core, and C6 refers todipentaerythritol (a hexaol) as die core. The “G-n” term refers to thegeneration number, which denotes the number of layers of branchingpoints which have been added, and which also refers to the number ofcoupling-and-deprotection iterations that have taken place. For example,“[G-1]” denotes “generation one,” and indicates that one round ofcoupling has occurred (see, e.g., dendrimer 1 of FIG. 1:“C3-([G-1]Ph)₃”) or that one round of coupling-and-deprotection hasoccurred (see, e.g., dendrimer 2 of FIG. 1: “C3-([G-1]OH₂)₃”). In otherwords, dendrimers 1 and 2 are of the same generation: generation one, or“G-1”. Each of the initiating alcohols bears a wedge shaped dendriticmoiety, referred to as a “dendron.” The end groups (per dendron) arenoted by either Ph_(p), for the benzylidene protected structures (where“p” has a value of 2^(n-1)), or OH_(q), for the hydroxylated structures(where “q” has a value of 2), and where “p” and “q” denote the number ofthe end groups per dendron (per wedge-shaped dendritic moiety). Finally,the number of dendrons per core, which corresponds to the core number,is denoted by “z”.

Example 1

General Synthetic Procedure

The general procedure for the preparation of the dendritic calibrantsfollows generally those published by Grayson et al. (Grayson, S. M.;Fréchet, J. M. J. Macromolecules, 2001; 34:6542-6544) and by Ihre et al.(Ihre, H.; Padilla de Jesus, O. L.; Fréchet, J. M. J J. Am. Chem. Soc.2001; 123:5908-5917), each of which are hereby incorporated by referencein their entireties.

As shown in FIG. 1, the dendritic synthesis involves the repetition oftwo critical steps: i) the dendritic growth or “dendronization” step, inwhich a “protected” monomer is attached to every active peripheralfunctionality; and ii) the activation or “deprotection” step, in whicheach monomer is altered to expose an increased multiplicity of activefunctionalities on the surface. Serial repetitions of these two stepslead to the exponential increase in both peripheral functional groupsand molecular weight.

Example 2 Preparation of Benzylidene Protected Bis-MPA Anhydride Monomer

The benzylidene protected bis-MPA anhydride monomer was preparedaccording to the synthesis reported previously by Ihre, H.; Padilla deJesus, O. L.; Fréchet, J. M. J J. Am. Chem. Soc. 2001, 123, 5908-5917,which is hereby incorporated by reference in its entirety.

Example 3

General Dendronization Procedure for Preparation of CX-([G-n]Ph_(p))_(z)

The procedure of this EXAMPLE is shown schematically as step “i” of FIG.1 (e.g., the syntheses of: dendrimer 1 from hydroxyl-terminated core; ofdendrimer 3 from dendrimer 2; etc.). To a round bottom flask were added:a known quantity of either hydroxyl-terminated core (e.g.,1,1,1-tris(hydroxymethyl)ethane, pentaerythritol, xylitol, ordipentaerythritol) or of dendrimer (e.g., one having the general formulaCX-([G-(n−1)]OH_(r))_(z), where “r” has a value of 2^((n-1)), asappropriate; 1.1 equivalents (per —OH of hydroxyl-terminated core or ofdendrimer) of the benzylidene protected bis-MPA anhydride monomer(bis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydridemonomer); and 0.1 molar equivalents (per —OH of hydroxyl-terminated coreor of dendrimer) of 4-dimethylaminopyridine (DMAP). The reaction mixturewas dissolved in the minimum amount of pyridine, diluted in twice thatamount (relative to pyridine) of dichloromethane, and the reactionmixture was then stirred vigorously for 4 hours at standard temperatureand pressure. The reaction was monitored periodically by MALDI-TOF MS todetermine the degree of coupling. After complete esterification wasobserved by MALDI-TOF MS, the flask contents were transferred to aseparatory funnel, diluted with dichloromethane, extracted three timeswith 1M aqueous NaHSO₄ (sodium bis sulfate) and three extractions with1M aqueous NaHCO₃ (sodium bicarbonate). The organic layers were reducedin vacuo to concentrate the sample, precipitated into hexanes, andfiltered to yield the benzylidene protected dendrimers,CX-([G-n]Ph_(p))_(z), as a white powdery precipitate. The resultingprecipitate may then be prepared for spectrometric analysis via standardprotocols.

Example 4

General Deprotection Procedure for Preparation of CX-([G-n]OH_(q))_(z)

The procedure of this EXAMPLE is shown schematically as step “ii” ofFIG. 1 (e.g., the syntheses of: dendrimer 2 from dendrimer 1; ofdendrimer 4 from dendrimer 3; etc.). To a round bottom flask, a measuredquantity of CX-([G-n]Ph_(r))_(z), where “r” has a value of 2^((n-1)) wasadded and dissolved in a sufficient amount of a 2:1 solution ofdichloromethane:methanol. Pearlman's catalyst (Pd(OH)₂/C) was added tothe reaction mixture, and the flask contents were placed under 8atmospheres (atm) of hydrogen gas. The reaction mixture was stirredvigorously for 24 hours at room temperature. Full deprotection wasverified by crude MALDI MS data, after which the Pd(OH)₂/C was removedvia filtration over Celite®. The filtrate was then reduced in vacuo toyield a transparent glassy solid having the formulaCX-([G-n]OH_(q))_(z). The resulting filtrate may then be prepared forspectrometric analysis via standard protocols.

Example 5

Synthesis of Tri-Functional “C-3” Calibrants

The tri-functional dendrimer species of this EXAMPLE 5 are shown in FIG.1.

Synthesis of C3-([G-1]Ph)₃, dendrimer 1 of FIG. 1:1,1,1-tris(hydroxymethyl)ethane (IUPAC name:2-(hydroxymethyl)-2-methylpropane-1,3-diol), which is commerciallyavailable, was esterified following the General Dendronization Procedureof EXAMPLE 3, using the benzylidene-protected Bis-MPA anhydride ofEXAMPLE 2 and DMAP to afford C3-([G-1]Ph)₃. Molecular Formula:C₄₁H₄₈O₁₂. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺ m/z=839.220.Observed MW: [M+Ag]⁺ m/z=839.20.

Synthesis of C3-([G-1]OH₂)₃, dendrimer 2 of FIG. 1: The benzylideneprotected dendrimer 1 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC3-([G-1]OH₂)₃. Molecular Formula: C₂₀H₃₆O₁₂. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=491.210. Observed MW: [M+Na]⁺ m/z=491.22.

Synthesis of C3-([G-2]Ph₂)₃, dendrimer 3 of FIG. 1: The hydroxylateddendrimer 2, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-2]Ph₂)₃. MolecularFormula: C₉₂H₁₀₈O₃₀. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=1799.598. Observed MW: [M+Ag]⁺ m/z=1799.59

Synthesis of C3-([G-2]OH₄)₃, dendrimer 4 of FIG. 1: The benzylideneprotected dendrimer 3 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC3-([G-2]OH₄)₃. Molecular Formula: C₅₀H₈₄O₃₀. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=1187.495. Observed MW: [M+Na]⁺ m/z=1187.46.

Synthesis of C3-([G-3]Ph₄)₃, dendrimer 5 of FIG. 1: The hydroxylateddendrimer 4, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-3]Ph₄)₃. MolecularFormula: C₁₉₄H₂₂₈O₆₆. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=3720.354. Observed MW: [M+Ag]⁺ m/z=3720.42.

Synthesis of C3-([G-3]OH₈)₃, dendrimer 6 of FIG. 1: The benzylideneprotected dendrimer 5 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC3-([G-3]OH₈)₃. Molecular Formula: C₁₁₀H₁₈₀O₆₆. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=2580.063. Observed MW: [M+Na]⁺m/z=2580.10.

Synthesis of C3-([G-4]Ph₈)₃, dendrimer 7 of FIG. 1: The hydroxylateddendrimer 6, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-4]Ph₈)₃. MolecularFormula: C₃₉₈H₆₈O₁₃₈. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=7561.865. Observed MW: [M+Ag]⁺ m/z=7559.9.

Synthesis of C3-([G-4]OH₁₆)₃, dendrimer 8 of FIG. 1: The benzylideneprotected dendrimer 7 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C3-([G-4]OH₁₆)₃. Molecular Formula: C₂₃₀H₃₇₂O₁₃₈. MALDI-TOFMS: Theoretical Exact MW: [M+Na]⁺ m/z=5365.256. Observed MW: [M+Na]⁺m/z=5366.6.

Synthesis of C3-([G-5]Ph₁₆)₃, dendrimer 9 of FIG. 1: The hydroxylateddendrimer 8, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-5]Ph₁₆)₃. MolecularFormula: C₈₀₆H₉₄₈O₂₈₂. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺ m/z=15256.1.Observed MW: [M+Ag]⁺ m/z=to be determined.

Synthesis of C3-([G-5]OH₃₂)₃, dendrimer 10 of FIG. 1: The benzylideneprotected dendrimer 9 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C3-([G-5]OH₃₂)₃. Molecular Formula: C₄₇₀H₇₅₆O₂₈₂. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=10942.0. Observed MW: [M+Na]⁺ m/z=to bedetermined.

Example 6

Synthesis of Tetra-Functional “C-4” Calibrants

The tetra-functional dendrimer species of this EXAMPLE 6 are shown inFIG. 2.

Synthesis of C4-([G-1]Ph)₄, dendrimer 11 of FIG. 2: Pentaerythritol(IUPAC name: 2,2-bis(hydroxymethyl)propane-1,3-diol), which iscommercially available, was esterified following the GeneralDendronization Procedure of EXAMPLE 3, using the benzylidene-protectedBis-MPA anhydride of EXAMPLE 2 and DMAP to afford C4-([G-1]Ph)₄.Molecular Formula: C₅₃H₆₀O₁₆. MALDI-TOF MS: Theoretical Exact MW:[M+Ag]⁺ m/z=1059.292. Observed MW: [M+Ag]⁺ m/z=1059.28.

Synthesis of C4-([G-1]OH₂)₄, dendrimer 12 of FIG. 2: The benzylideneprotected dendrimer 11 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC4-([G-1]OH₂)₄. Molecular Formula: C₂₅H₄₄O₁₆. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=623.252. Observed MW: [M+Na]⁺ m/z=623.05.

Synthesis of C4-([G-2]Ph₂)₄, dendrimer 13 of FIG. 2: The hydroxylateddendrimer 12, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C4-([G-2]Ph₂)₄. MolecularFormula: C₁₂₁H₁₄₀O₄₀. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=2339.797. Observed MW: [M+Ag]⁺ m/z=2339.85.

Synthesis of C4-([G-2]OH₄)₄, dendrimer 14 of FIG. 2: The benzylideneprotected dendrimer 13 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4 to affordC4-([G-2]OH₄)₄. Molecular Formula: C₆₅H₁₀₈O₄₀. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=1551.631. Observed MW: [M+Na]⁺ m/z=1551.62.

Synthesis of C4-([G-3]Ph₄)₄, dendrimer 15 of FIG. 2: The hydroxylateddendrimer 14, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C4-([G-3]Ph₄)₄. MolecularFormula: C₂₅₇H₃₀₀O₈₈. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=4900.805. Observed MW: [M+Ag]⁺ m/z=4900.98

Synthesis of C4-([G-3]OH₈)₄, dendrimer 16 of FIG. 2: The benzylideneprotected dendrimer 15 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC4-([G-3]OH₈)₄. Molecular Formula: C₁₄₅H₂₃₆O₈₈. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=3408.389 Observed MW: [M+Na]⁺m/z=3408.41.

Synthesis of C4-([G-4]Ph₈)₄, dendrimer 17 of FIG. 2: The hydroxylateddendrimer 16, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-4]Ph₈)₄. MolecularFormula: C₅₂₉H₆₂₀O₁₈₄. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺ m/z=10030.5Observed MW: [M+Ag]⁺ m/z=10018.1.

Synthesis of C4-([G-4]OH₁₆)₄, dendrimer 18 of FIG. 2: The benzylideneprotected dendrimer 17 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C4-([G-4]OH₁₆)₄. Molecular Formula: C₃₀₅H₄₉₂O₁₈₄. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=7126.1. Observed MW: [M+Na]⁺ m/z=7123.5.

Synthesis of C4-([G-5]Ph₁₆)₄, dendrimer 19 of FIG. 2: The hydroxylateddendrimer 18, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C4-([G-5]Ph₁₆)₄. MolecularFormula: C₁₀₇₃H₁₂₆₀O₃₇₆. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺m/z=20281.4. Observed MW: [M+Ag]⁺ m/z=to be determined.

Synthesis of C4-([G-5]OH₃₂)₄, dendrimer 20 of FIG. 2: The benzylideneprotected dendrimer 19 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C4-([G-5]OH₃₂)₄. Molecular Formula: C₆₂₅H₁₀₀₄O₃₇₆. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=14557.6. Observed MW: [M+Na]⁺ m/z=to bedetermined.

Example 7

Synthesis of Penta-Functional “C-5” Calibrants

The penta-functional dendrimer species of this EXAMPLE 7 are shown inFIG. 3.

Synthesis of C5-([G-1]Ph)₅, dendrimer 21 of FIG. 3: Xylitol (IUPAC name:pentane-1,2,3,4,5-pentol), which is commercially available, wasesterified following the General Dendronization Procedure of EXAMPLE 3,using the benzylidene-protected Bis-MPA anhydride of EXAMPLE 2 and DMAPto afford C5-([G-1]Ph)₅. Molecular Formula: C₆₅H₇₂O₂₀. MALDI-TOF MS:Theoretical Exact MW: [M+Ag]⁺ m/z=1279.366. Observed MW: [M+Ag]⁺m/z=1279.39.

Synthesis of C5-([G-1]OH₂)₅, dendrimer 22 of FIG. 3: The benzylideneprotected dendrimer 21 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC5-([G-1]OH₂)₅. Molecular Formula: C₃₀H₅₂O₂₀. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=755.295. Observed MW: [M+Na]⁺ m/z=755.17.

Synthesis of C5-([G-2]Ph₂)₅, dendrimer 23 of FIG. 3: The hydroxylateddendrimer 22, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C5-([G-2]Ph₂)₅. MolecularFormula: C₁₅₀H₁₇₂O₅₀. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=2879.997. Observed MW: [M+Ag]⁺ m/z=2880.01.

Synthesis of C5-([G-2]OH₄)₅, dendrimer 24 of FIG. 3: The benzylideneprotected dendrimer 23 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC5-([G-2]OH₄)₅. Molecular Formula: C₈₀H₁₃₂O₅₀. Molecular Formula:C₁₅₀H₁₇₂O₅₀. MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=1915.768.Observed MW: [M+Na]⁺ m/z=1915.78.

Synthesis of C5-([G-3]Ph₄)₅, dendrimer 25 of FIG. 3: The hydroxylateddendrimer 24, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C5-([G-3]Ph₄)₅. MolecularFormula: C₃₂₀H₃₇₂O₁₁₀. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=6081.257. Observed MW: [M+Ag]⁺ m/z=6081.51.

Synthesis of C5-([G-3]OH), dendrimer 26 of FIG. 3: The benzylideneprotected dendrimer 25 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC5-([G-3]OH₈)₅. Molecular Formula: C₁₈₀H₂₉₂O₁₁₀. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=4236.715. Observed MW: [M+Na]⁺m/z=4236.80.

Synthesis of C5-([G-4]Ph₈)₅, dendrimer 27 of FIG. 3: The hydroxylateddendrimer 26, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C5-([G-4]Ph₈)₅. MolecularFormula: C₆₆₀H₇₇₂O₂₃₀. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺ m/z=12493.1.Observed MW: [M+Ag]⁺ m/z=12476.0.

Synthesis of C5-([G-4]OH₁₆)₅, dendrimer 28 of FIG. 3: The benzylideneprotected dendrimer 27 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C5-([G-4]OH₁₆)₅. Molecular Formula: C₃₈₀H₆₁₂O₂₃₀. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=8883.9. Observed MW: [M+Na]⁺ m/z=8880.1.

Synthesis of C5-([G-5]Ph₁₆)₅, dendrimer 29 of FIG. 3: The hydroxylateddendrimer 28, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C5-([G-5]Ph₁₆)₅. MolecularFormula: C₁₃₄₀H₁₅₇₂O₄₇₀. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺m/z=25306.7. Observed MW: [M+Ag]⁺ m/z=to be determined.

Synthesis of C5-([G-5]OH₃₂)₅, dendrimer 30 of FIG. 3: The benzylideneprotected dendrimer 29 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C5-([G-5]OH₃₂)₅. Molecular Formula: C₇₈₀H₁₂₅₂O₄₇₀. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=18173.2. Observed MW: [M+Na]⁺ m/z=to bedetermined.

Example 8

Synthesis of Hexa-Functional “C-6” Calibrants

The hexa-functional dendrimer species of this EXAMPLE 8 are shown inFIG. 4.

Synthesis of C6-([G-1]Ph)₆, dendrimer 31 of FIG. 4: Dipentaerythritol(IUPAC name:2-[[3-hydroxy2,2-bis(hydroxymethyl)propoxy]methyl]-2-(hydroxymethyl)propane-1,3-diol),which is commercially available, was esterified following the GeneralDendronization Procedure of EXAMPLE 3, using the benzylidene-protectedBis-MPA anhydride of EXAMPLE 2 and DMAP to afford C6-([G-1]Ph)₆.Molecular Formula: C₈₂H₉₄O₂₅. MALDI-TOF MS: Theoretical Exact MW:[M+Ag]⁺ m/z=1585.514. Observed MW: [M+Ag]⁺ m/z=1585.53.

Synthesis of C6-([G-1]OH₂)₆, dendrimer 32 of FIG. 4: The benzylideneprotected dendrimer 31 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC6-([G-1]OH₂)₆. Molecular Formula: C₄₀H₇₀O₂₅. MALDI-TOF MS: TheoreticalExact MW: [M+Na]⁺ m/z=973.410. Observed MW: [M+Na]⁺ m/z=973.34.

Synthesis of C6-([G-2]Ph₂)₆, dendrimer 33 of FIG. 4: The hydroxylateddendrimer 32, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C6-([G-2]Ph₂)₆. MolecularFormula: C₁₈₄H₂₁₄O₆₁. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=3506.269. Observed MW: [M+Ag]⁺ m/z=3506.25.

Synthesis of C6-([G-2]OH₄)₆, dendrimer 34 of FIG. 4: The benzylideneprotected dendrimer 33 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC6-([G-2]OH₄)₆. Molecular Formula: C₁₀₀H₁₆₆O61. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=2365.979. Observed MW: [M+Na]⁺m/z=2365.98.

Synthesis of C6-([G-3]Ph₄)₆, dendrimer 35 of FIG. 4: The hydroxylateddendrimer 34, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C6-([G-3]Ph₄)₆. MolecularFormula: C₃₈₈H₄₅₄O₁₃₃. MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=7347.781. Observed MW: [M+Ag]⁺ m/z=7347.0.

Synthesis of C6-([G-3]OH₈)₆, dendrimer 36 of FIG. 4: The benzylideneprotected dendrimer 35 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC6-([G-3]OH₈)₆. Molecular Formula: C₂₂₀H₃₅₈O₁₃₃. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=5151.115. Observed MW: [M+Na]⁺m/z=5151.28.

Synthesis of C6-([G-4]Ph₈)₆, dendrimer 37 of FIG. 4: The hydroxylateddendrimer 36, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C6-([G-4]Ph₈)₆. MolecularFormula: C₇₉₆H₉₃₄O₂₇₇. MALDI-TOF MS: Theo. Avg. MW: [M+Ag]⁺ m/z=14969.7.Observed MW: [M+Ag]⁺ m/z=15020.1.

Synthesis of C6-([G-4]OH₁₆)₆, dendrimer 38 of FIG. 4: The benzylideneprotected dendrimer 37 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C6-([G-4]OH₁₆)₆. Molecular Formula: C₄₆₀H₇₄₂O₂₇₇. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=10655.6. Observed MW: [M+Na]⁺m/z=10722.6.

Synthesis of C6-([G-5]Ph₁₆)₆, dendrimer 39 of FIG. 4: The hydroxylateddendrimer 38, would be esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C6-([G-5]Ph₁₆)₆. MolecularFormula: C₁₆₁₂H₁₈₉₄O₅₆₅. MALDI-TOF MS: Theo. Avg. MW: [+Ag]⁺m/z=30346.1. Observed MW: [M+Ag]⁺ m/z=to be determined.

Synthesis of C6-([G-5]OH₃₂)₆, dendrimer 40 of FIG. 4: The benzylideneprotected dendrimer 39 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford C6-([G-5]OH₃)₆. Molecular Formula: C₉₄₀H₁₅₁₀O₅₆₅. MALDI-TOFMS: Theo. Avg. MW: [M+Na]⁺ m/z=21802.8. Observed MW: [M+Na]⁺ m/z=to bedetermined.

Example 9

Parallel Synthesis of Dendrimers 1, 11, 21, and 31

In the prior art, a broad range calibrant is made by mixing appropriatequantities of individual peptides, which have been prepared and purifiedseparately, to yield a calibrant cocktail. The synthetic methodologydescribed herein and shown schematically in FIG. 5, however, provides aunique way to prepare calibrant sets by starting with a mixture ofwell-defined commercially available starting materials, and dendronizingthem in parallel.

By serial repetitions of steps “i” and “ii” as detailed in EXAMPLES 3and 4 (and as shown, for example, in FIG. 1), dendrimers can be preparedwith (approximately) exponentially increasing molecular weights. Forexample, by starting with just the C-3 hydroxyl-terminated core, serialrepetition of steps “i” and “ii” can produce monodisperse dendrimercalibrants (e.g., dendrimers 1, 3, 5, 7, 9, etc. of FIG. 1) that haveapproximate molecular weights of 730, 1690, 3610, 7450, 15100, and30500. By starting with a different core, bearing a different number ofalcohol functionalities, (e.g., the C-4, C-5, or C-6 hydroxyl-terminatedcore), a wide range of calibrants with a broad distribution can beefficiently prepared.

A particularly efficient way to make a calibrant mixture is to carry outthe dendronization process using a mixture of cores in a single batch(e.g., equimolar mixtures of the C-3, C-4, C-5, and/or the C-6 cores).For example, and as shown in FIG. 5, after a single dendronization step,the mixture of four cores will yield a set of “first generation”dendrimers 1, 11, 21, and 31 having molecular weights (with silvercounterion) of 839, 1059, 1279, and 1585 (as demonstrated in FIG. 6).After an additional repetition of steps “ii” and “i,” also shown in FIG.5, the set of “second generation” calibrants (3, 13, 23, 33) havemolecular weights of 1800, 2340, 2880, and 3506 (as demonstrated in FIG.7). In this way, serial repetitions of steps “i” and “ii” enable rapidaccess to a series of 4-point sets (see, e.g., FIGS. 6-12).

Because the most desirable calibrant would be a mixture of numerous,well-defined monodisperse compounds (e.g., as shown in the reactionscheme of FIG. 5 and the spectra of FIGS. 6-12), this describedsynthetic technique has the additional advantage that the differentcalibrants can be prepared together in one batch (by dendronizing aselected mixture of cores), rather than preparing each speciesseparately and mixing them after the isolating of each product. Becauseprevious attempts to prepare dendrimers sought a well-defined singularproduct, this parallel approach is both unprecedented and valuable inreducing the cost and effort of preparing sets of calibrants.

Synthesis of CX-([G-1]Ph)_(z), an equimolar mixture of dendrimers 1, 11,21, and 31 (see, e.g., reaction scheme of FIG. 5): An equimolar mixtureof (trishydroxymethyl)ethane (C3-OH₃), pentaerythritol (C4-OH₄), xylitol(C5-OH₅), and dipentaerythritol (C6-OH₆) was esterified following theGeneral Dendronization Procedure of EXAMPLE 3, using thebenzylidene-protected Bis-MPA anhydride of EXAMPLE 2 and DMAP to affordthe CX-([G-1]Ph), mixture of dendrimers 1, 11, 21, and 31. As shown inFIG. 6, MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺ m/z=839.220;1,059.293; 1,279.367; 1,585.514. Observed MW: [M+Ag]⁺ m/z=839.20;1,059.28; 1,279.39; 1585.53. As can be appreciated from FIG. 6, themixture of dendrimers 1, 11, 21, and 31 provides an effective four-pointcalibration that covers the 800-1,600 mass range.

Example 10

Parallel Synthesis of Dendrimers 2, 12, 22, and 32

Synthesis of CX-([G-1]OH₂)_(z), an equimolar mixture of dendrimers 2,12, 22, and 32 (not shown) (see, e.g., reaction scheme of FIG. 5): Themixture of benzylidene protected dendrimers 1, 11, 21, and 31 fromEXAMPLE 9 was deprotected using 5% Pd(OH)₂/C and hydrogen gas followingthe General Deprotection Procedure of EXAMPLE 4, to afford theCX-([G-1]OH₂)_(z), mixture of dendrimers 2, 12, 22, and 32. MALDI-TOFMS: Theoretical Exact MW: [M+Na]⁺ m/z=491.210; 623.253; 755.295;973.410. Observed MW: [M+Na]⁺ m/z=491.22; 623.05; 755.17; and 973.34.

Example 11

Parallel Synthesis of Dendrimers 3, 13, 23, and 33

Synthesis of CX-([G-2]Ph₂)_(z), an equimolar mixture of dendrimers 3,13, 23, and 33 (see, e.g., reaction scheme of FIG. 5): The mixture ofhydroxyl functionalized dendrimers 2, 12, 22, and 32 from EXAMPLE 10 wasesterified following the General Dendronization Procedure of EXAMPLE 3,using the benzylidene-protected Bis-MPA anhydride of EXAMPLE 2 and DMAPto afford the CX-([G2]Ph₂)_(z), mixture of dendrimers 3, 13, 23, and 33.As shown in FIG. 7, MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=1,799.598; 2,339.797; 2,879.997; 3,506.269. Observed MW: [M+Ag]⁺m/z=1,799.59; 2,339.85; 2,880.01; 3,506.25. As can be appreciated fromFIG. 7, the mixture of dendrimers 3, 13, 23, and 33 provides aneffective four point calibration that covers the 1,800-3,600 mass range.

Example 12

Parallel Synthesis of Dendrimers 4, 14, 24, and 34

Synthesis of CX-([G-2]OH₄)_(z), an equimolar mixture of dendrimers 4,14, 24, and 34 (see, e.g., reaction scheme of FIG. 5): The mixture ofbenzylidene protected dendrimers 3, 13, 23, and 33 from EXAMPLE 11 wasdeprotected using 5% Pd(OH)₂/C and hydrogen gas following the GeneralDeprotection Procedure of EXAMPLE 4, to afford the CX-([G-2]OH₄)_(z)mixture of dendrimers 4, 14, 24, and 34. As shown in FIG. 8, MALDI-TOFMS: Theoretical Exact MW: [M+Na]⁺ m/z=1,187.495; 1,551.631; 1,915.768;2,365.979. Observed MW: [M+Na]⁺ m/z=1,187.46; 1,551.62; 1,915.78;2,365.98. As can be appreciated from FIG. 7, the mixture of dendrimers4, 14, 24, and 34 provides an effective four point calibration thatcovers the 1,200-2,400 mass range.

Example 13

Parallel Synthesis of Dendrimers 5, 15, 25, and 35

Synthesis of CX-([G-3]Ph₄)_(z), an equimolar mixture of dendrimers 5,15, 25, and 35 (see, e.g., reaction scheme of FIG. 5): The mixture ofhydroxyl functionalized dendrimers 4, 14, 24, and 34 from EXAMPLE 12 wasesterified following the General Dendronization Procedure of EXAMPLE 3,using the benzylidene-protected Bis-MPA anhydride from EXAMPLE 2 andDMAP to afford the CX-([G3]Ph₄)_(z), mixture of dendrimers 5, 15, 25,and 35. As shown in FIG. 9, MALDI-TOF MS: Theoretical Exact MW: [M+Ag]⁺m/z=3,720.354; 4,900.805; 6,081.257; 7,347.781. Observed MW: [M+Ag]⁺m/z=3,720.42; 4,900.98; 6,081.51; and 7,348.00. As can be appreciatedfrom FIG. 9, the mixture of dendrimers 5, 15, 25, and 35 provides aneffective four point calibration that covers the 3,600-7,200 mass range.

Example 14

Parallel Synthesis of Dendrimers 6, 16, 26, and 36

Synthesis of CX-([G-3]OH₈)_(z), an equimolar mixture of dendrimers 6,16, 26, 36 (see, e.g., reaction scheme of FIG. 5): The mixture ofbenzylidene protected dendrimers 5, 15, 25, and 35 from EXAMPLE 13 wasdeprotected using 5% Pd(OH)₂/C and hydrogen gas following the GeneralDeprotection Procedure of EXAMPLE 4, to afford the CX-([G-3]OH₈)_(z)mixture of dendrimers 6, 16, 26, 36. As shown in FIG. 10, MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=2,580.063; 3,408.389; 4,236.715;5,151.115. Observed MW: [M+Na]⁺ m/z=2,580.10; 3,408.41; 4,236.80;5,151.28. As can be appreciated from FIG. 10, the mixture of dendrimers6, 16, 26, and 36 provides an effective four point calibration thatcovers the 2,500-5,100 mass range.

Example 15

Parallel Synthesis of Dendrimers 7, 17, 27, and 37

Synthesis of CX-([G-4]Ph₈)_(z), an equimolar mixture of dendrimers 7,17, 27, and 37 (see, e.g., reaction scheme of FIG. 5): The mixture ofhydroxyl functionalized dendrimers 6, 16, 26, and 36 from EXAMPLE 14 wasesterified following the General Dendronization Procedure of EXAMPLE 3,using the benzylidene-protected Bis-MPA anhydride, and DMAP to affordthe CX-([G-4]Ph₈)_(z) mixture of dendrimers 7, 17, 27, and 37. MALDI-TOFMS: Theo. Avg. MW: [M+Ag]⁺ m/z=7,561.9; 10,022.8; 12,483.8; 15,030.8.Observed MW: [M+Ag]⁺ m/z=7,562; 10,023; 12,484; 15,031. As can beappreciated from FIG. 11, the mixture of dendrimers 7, 17, 27, and 37provides an effective four point calibration that covers the7,500-15,000 mass range.

Example 16

Parallel Synthesis of Dendrimers 8, 18, 28, and 38

Synthesis of CX-([G-4]OH₁₆)_(z), an equimolar mixture of dendrimers 8,18, 28, and 38: The mixture of benzylidene protected dendrimers 7, 17,27, and 37 from EXAMPLE 15 was deprotected using 5% Pd(OH)₉/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford the CX-([G-4]OH₁₆)_(z), mixture of dendrimers 8, 18, 28, and38. MALDI-TOF MS: Theo. Avg. MW: [M+Na]⁺ m/z=5,365.2; 7,121.9; 8,878.6;10,721.4. Observed MW: [M+Na]⁺ m/z=5,366.619; 7,123.504; 8,880.111;10,722.572. As can be appreciated from FIG. 12, the mixture ofdendrimers 8, 18, 28, and 38 provides an effective four pointcalibration that covers the 5,500-10,500 mass range.

Example 17

Parallel Synthesis of Dendrimers 9, 19, 29, and 39

Synthesis of CX-([G-5]Ph₁₆)_(z), an equimolar mixture of dendrimers 9,19, 29, and 39: The mixture of hydroxyl functionalized dendrimers 8, 18,28, and 38 from EXAMPLE 16 would be esterified following the GeneralDendronization Procedure of EXAMPLE 3, using the benzylidene-protectedBis-MPA anhydride of EXAMPLE 3 and DMAP to afford the CX-([G-5]Ph₁₆)_(z)mixture of dendrimers 9, 19, 29, and 39. MALDI-TOF MS: Theo. Avg. MW:[M+Ag]⁺ m/z=15,244.9; 20,266.9; 25,288.8; 30,396.9. Observed MW: [M+Ag]⁺m/z=to be determined.

Example 18

Parallel Synthesis of Dendrimers 10, 20, 30, and 40

Synthesis of CX-([G-5]OH₃₂)_(z), an equimolar mixture of dendrimers 10,20, 30, and 40: The mixture of benzylidene protected dendrimers, 9, 19,29, and 39 from EXAMPLE 17 would be deprotected using 5% Pd(OH)₂/C andhydrogen gas following the General Deprotection Procedure of EXAMPLE 4,to afford the CX-([G-5]OH₃₂)_(z) mixture of dendrimers 10, 20, 30, and40. MALDI-TOF MS: Theo. Avg. MW: [M+Na]⁺ m/z=10,935.5; 14,548.9;18,162.4; 21,861.9. Observed MW: [M+Na]⁺ m/z=to be determined

Example 19

Calibrant Tests—Dendronized Cavitand

To verify the utility of the calibrants of the present disclosure inacquiring accurate MALDI-TOF data with high mass resolution, adendronized cavitand (a monodisperse synthetic molecule) was examined,and the results are shown in FIG. 13A. The dendronized cavitand(Cav-([G1]-Ph)₈, as shown in FIG. 13B) has the molecular formulaC₁₉₂H₁₇₆O₄₈, MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=3,272.122.Observed MW: [M+Na]⁺ m/z=3,272.06. Mass Accuracy: 18.9 ppm.

Example 20

Calibrant Test—Poly(ethylene) Glycol, PEG 1970

To further verify the utility of the calibrants of the presentdisclosure in acquiring accurate MALDI-TOF data with high massresolution, synthetic polymer PEG 1970 (a polydisperse polymer of threedifferent oligomers: a 33-mer, a 43-mer, and a 53 mer), was examined.The number average molecular weight (M_(n)) of PEG 1970 is 1970, and itspolydispersity index (PDI) is 1.05. The spectrometric results are shownin FIGS. 14-16.

The PEG 1970 33-mer has the molecular formula C₆₆H₁₃₄O₃₄. As shown inFIG. 14, MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=1493.865.Observed MW: [M+Na]⁺ m/z=1493.96. Mass Accuracy: 63.6 ppm.

The PEG 1970 43-mer has the molecular formula C₈₆H₁₇₄O₄₄. As shown inFIG. 15, MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=1934.127.Observed MW: [M+Na]⁺ m/z=1934.20. Mass Accuracy: 37.7 ppm.

The PEG 1970 53-mer has the molecular formula C₁₀₆H₂₁₄O₅₄. As shown inFIG. 16, MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=2374.389.Observed MW: [M+Na]⁺ m/z=2374.44. Mass Accuracy: 21.5 ppm.

Example 21

Calibrant Test—Proprietary Peptide JF-1485

To further verify the utility of the calibrants of the presentdisclosure in acquiring accurate MALDI-TOF data with high massresolution, peptide JF-1485 having the formula C₈₈H₁₁₈N₁₆O₂₂S₅ (andhaving a proprietary structure) was examined. As shown in FIG. 17,MALDI-TOF MS: Theoretical Exact MW of the H⁺ adduct: [M+H]⁺m/z=1911.728. Observed MW: [M+H]⁺ m/z=1911.68. Theoretical Exact MW ofthe Na⁺ adduct: [M+Na]⁺ m/z=1933.7102. Observed MW: [M+Na]⁺ m/z=1933.69.Theoretical Exact MW of the K⁺ adduct: [M+K]⁺ m/z=1949.6842. ObservedMW: [M+K]⁺ m/z=1949.60. Mass Accuracy: 25.1 ppm.

Alternative Hydroxyl-Terminated Cores

As will be appreciated by those having ordinary skill in the art,dendrimers of various functionalities other than the ones describedabove may be synthesized via the General Dendronization Procedure ofEXAMPLE 3 followed (optionally) by the General Deprotection Procedure ofEXAMPLE 4. This could be accomplished, for example, and withoutintending to be limited, simply by choosing a hydroxyl-terminated coredifferent from the ones disclosed above (e.g., a core other than1,1,1-tris(hydroxymethyl)ethane, pentaerythritol, xylitol, ordipentaerythritol) for the General Dendronization Procedure of EXAMPLE3. Exemplary alternative hydroxyl-terminated cores include, withoutintending to be limited: tripentaerythritol (eight hydroxyl termini) andtetrapentaerythritol (ten hydroxyl termini). Those having ordinary skillin the art will also understand from the foregoing description that eachdendrimer created via the General Dendronization Procedure of EXAMPLE 3may also function as an alternative hydroxyl-terminated core. Forexample, the dendrimer denoted C3-([G-2]OH₄)₃—dendrimer 4 of FIG.1—possesses twelve —OH termini, each of which may undergo a round ofdendronization (via the General Dendronization Procedure of EXAMPLE 3).The resulting dendrimer may then undergo the General DeprotectionProcedure of EXAMPLE 4 to yield yet another dendrimer, and the steps maybe repeated to create even larger dendrimers. Thus, alcohols containingfrom about 1 to many hundreds of hydroxyl (—OH) termini may be used inthe General Dendronization Procedure of EXAMPLE 3 (preferablypolyalcohols, and including linear polyols such as poly(vinyl alcohol)and hyperbranched polyols such as poly(glycerols)), and followed(optionally) by the General Deprotection Procedure of EXAMPLE 4 toproduce calibrants useful for mass spectrometry, especially forMALDI-TOF, ESI, APCI, and FAB techniques. Moreover, combinations of suchalcohols (and preferably polyalcohols) may be used in parallel syntheses(e.g., as described in EXAMPLES 9-18) to create a panel of calibrantsuseful across a broad range of m/z ratios.

In addition, the coupling acylation chemistry used to covert alcohols tothe corresponding esters during the “coupling” or “dendronization” stepas described in EXAMPLE 3 is equally amenable to the acylation reaction,using the same reagents, that converts amines to amides. As a result,polyamine core molecules can also be used (as core molecules), includingcommercially available families of dendritic polyamine such as thepoly(amidoamine) (PAMAM) and poly(propylene amine) (PPI) dendrimers.

Trismonomer

The benzylidene protected bis-MPA monomer described above may bemodified by substituting a hydroxymethyl group for the pendent methylgroup, to produce a protected trismonomer, as shown in Formula 1 below:

By substituting a hydroxymethyl group for the pendent methyl group ofthe benzylidene protected bis-MPA anhydride monomer((bis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydridemonomer), each dendrimer layer could contain three branches, rather thanthe two branches shown in FIGS. 1-5. In other words, by using themonomer of Formula 1 in the General Dendronization Procedure of EXAMPLE3 and subsequently in the General Deprotection Procedure of EXAMPLE 4,each branch point would yield three branches, instead of the twobranches shown in FIGS. 1-5. For example, by starting with1,1,1-tris(hydroxymethyl)ethane and using the trismonomer of Formula 1for one round of dendronization and deprotection according to EXAMPLES 3and 4, respectively, a C3 calibrant —C3-([G-1]OH₃)₃—according to Formula2 (and similar to dendrimer 2) would be produced:

The —OH groups of Formula 2 may be protected using methylideneorthoesters to carry out subsequent dendronization and deprotectionsteps.

Tuning the Dendrimers

Because the dendrimers described originate almost exclusively from thebis(hydroxymethyl)propanoic acid monomer, the composition of the overallstructure can be easily tuned by subtle changes in the monomerstructure. Such tuning could include modification of a pendant methylgroup and/or synthesis of dendrimers using ¹²C isotopically-enrichedmonomer.

The exact atomic masses of all atoms are close to, but not exactly,whole numbers. Because larger molecular weight (MW) compounds arecomprised of multiple atoms, they have a significant mass defect—anoffset from the nominal mass (the value of the nearest integerapproximation of the most abundant isotope for each atomic mass). Simplyput, the mass defect is the difference between the whole numberapproximate “nominal mass” and the actually-observed monoisotopic mass.The mass defect can be used to identify classes of compounds, and can beused to distinguish natural biomolecules from unnaturally modified ones.By tuning the elemental composition of the dendrimer backbone, the massdefect can be adjusted to ensure that they do not overlap with—and canbe easily differentiated from—natural compounds. Such tuning can alsofacilitate automated data analysis by simplifying the distinctionbetween analyte and calibrant. Because the disclosed dendrimers are madepredominantly by multiple layers of the same monomer, tuning theelemental composition of that monomer allows the mass defect of all ofthe disclosed dendrimers to be tuned. For example, an average peptidewill exhibit the “averagine” mass defect of +0.506 daltons (Da) per 1000Da of molecular weight. “Averagine” is the theoretical “average” aminoacid in regards to its elemental composition (with the non integermolecular formula: C_(4.9384)H_(7.7583)N_(1.3577)O_(1.4773) S_(0.0417)),and can be used to calculate the expected elemental composition and massdefect of peptides and proteins across a range of molecular weights. Thehydroxyl-functionalized dendrons (see, e.g., dendrimers 2, 4, 6, 8,etc.) exhibit a mass defect of +0.42±0.02 Da per 1000 Da of molecularweight, while the benzylidene functionalized dendrons (see, e.g.,dendrimers 1, 3, 5, 7, 9, etc.) exhibit a mass defect of 0.39±0.02 Daper 1000 Da of molecular weight. In order to differentiate this massdefect further, the pendant methyl of the hydroxyl-functionalizeddendrons can be modified or functionalized with a variety of longeralkyl chains or with halogenated alkyl chains, without any significanteffect on the synthetic procedure. This may be accomplished by modifyingthe benzylidene protected bis-MPA anhydride monomer(bis(5-methyl-2-phenyl-1,3-dioxane-5-carboxylic) acid anhydride monomerat the 5-methyl position as shown in Formula 3 below:

In Formula 3, X may be: alkyl (e.g., CH₃, CH₂CH₃, CH₂CH₂CH₃, or(CH₂)_(n)CH₃, where n is an integer from 0 to 16); CH₂—O—CH₂-Ph, wherePh represents phenyl; CQ₃, where “Q” represents a halogen, preferablyfluorine (F) or chlorine (Cl) (e.g., CF₃, CCl₃, etc.); or (CQ₂)_(n)CQ₃,where “Q” represents a halogen, preferably fluorine (F) or chlorine(Cl), and where n is an integer from 1 to 16. For example, a rathersignificant shift in MW can be demonstrated by replacing the methylgroup with a trifluoromethyl group, resulting in a shift in the massdefect to +0.11±0.02 Da per 1000 Da of MW. The molecular mass defect canalso be modified by a simple functionalization of the periphery with asubstituent with the desired mass defect. Despite modification at “X,”dendrimer synthesis using the benzylidene protected monomer of Formula 3may proceed via serial iterations of the General DendronizationProcedure of EXAMPLE 3 and the General Deprotection Procedure of EXAMPLE4.

As the molecular weight of carbon-containing molecules increases, thenatural prevalence of ¹³C (natural abundance=1.109%) in the moleculesleads to a broadening of the molecular isotopic distribution in theirmass spectra. Above about 8,000 Da, the signal corresponding to themonoisotopic species (having only ¹²C) is so small, relative topolyisotopic species, that exact mass determination is difficult becausethe monoisotopic species' peak is difficult to identify amongst thepeaks from polyisotopic species. Consequently, the presence ofpolyisotopic species greatly reduces the resolution of molecular weightcalculations. Take, for example, Formula 4:

which can be represented by the formula C₅H₁₂O₄. Because greater than 1%of C is ¹³C, the MS of any carbon-containing compound will exhibithigher molecular weight signals corresponding to these ¹³C isotopes. Asthe number of carbons in a compound increases, the likelihood that ¹³Cis present in the compound increases. This is seen in the isotopicdistribution of the monomer of Formula 4, which has an exact mass of136.07356, exhibits a monoisotopic signal at 136.07356 (m/z; 100.0%relative signal intensity), and a higher molecular weight species at137.07691 (m/z; 5.4% relative signal intensity).

With increasing carbon content (e.g., without intending to be limited,500 carbon atoms per molecule) the statistical distribution of molecularweights from different polyisotopic species becomes so broad that thesingle monoisotopic peak can become difficult to resolve. The nativeabundance of ¹²C is 98.89%, of ¹³C is 1.109%, of ¹H is 99.99%, of ²H is0.01%, of ¹⁶O is 99.76%, of ¹⁸O is 0.20%, and of ¹⁷O is 0.04%. The ¹³Cisotope is the most common higher isotope in most organic compounds.Thus, the simplest way to narrow the isotopic distribution at highmolecular weights is to start with building materials in which ¹³C hasbeen depleted—for example, starting materials in which all carbon is¹²C.

Because the dendrimers described originate almost exclusively from thebis(hydroxymethyl)propanoic acid monomer, if the synthesis is carriedout with ¹²C isotopically enriched monomer then the mass spectral peakbroadening will be reduced substantially, and high accuracy calibrationabove 10,000 Da can be achieved easily. While isotopic broadening due to¹⁸O is much less pronounced (because 180 represents only 0.201% of all Ospecies) ¹⁶O isotopic enrichment can also be carried out to improve theaccuracy even further. These isotopic enrichments contemplated here arenot expected to have any effect on the synthetic parameters, beyondsubtly altering the molecular weights of the reactants and the dendrimerproducts.

As shown in the General Dendronization Procedure for Preparation ofCX-([G-n]Ph_(p))_(z) described in EXAMPLE 3, the alcohol functionalitiesof the monomer must be “protected” in order to control the iterativedendrimer growth that yields exact monodisperse structures. Two alcoholscan be protected simultaneously with benzylidene (described in EXAMPLE 3and shown below at Formula 5), and those of ordinary skill in the artwill also recognize that they may be protected with acetonide (Formula6), or other acetal or ketal protecting group (see, e.g., Formulae 7 &8, where R³ is H or CHA, R⁴ is Ph, CH₃, C₆H₄OCH₃, or C₆H₄NO₂, R₅ isCH₂Ph, Si(CH₃)₃, C₆H₅NO₂, CH₂OCH₃, C₅H₉O (Tetrahydropyranyl ether), orSiPh₂t-Bu, and where Ph is phenyl).

Further examples of protecting groups may be found in “Protective Groupsin Organic Synthesis” by P. G. M. Wuts and T. W. Greene (4th edition,2007, John Wiley and Sons Inc. Hoboken, N.J.), which is incorporated byreference herein in its entirety. In addition, a number of labile etherlinkages, including benzyl ethers, substituted benzyl ethers, and silylethers, can be also be used instead of, or in addition to, to enable thesynthesis of structurally pure dendrimers. Such modifications to thedendronization procedure lie within the scope of the present disclosure.

Tuning the Dendrimers Via the Core Molecule

Another method of tuning is to modify the core molecule of thedendrimer. In one embodiment, the dendrimer is tuned so as toincorporate a specific element or elements not commonly found inbiomolecules into the core molecule in order to create a dendriticcalibrant with a mass defect marker distinct from common, naturalbiomolecules. By tuning the elemental composition of the dendrimer coremolecule, the mass defect can be adjusted to ensure that the observedmasses of the dendrimers do not overlap with—and therefore can be easilydifferentiated from—the more common natural compounds during massspectrometry.

Fluorine, phosphorus and iodine are all speculated to be desirableelements for incorporation into the core molecule because they arebelieved to result in a comparatively stable dendrimer calibrant thatfurther results in a mass spectral peak of a narrower width.Specifically, because halogens (e.g., fluorine, chlorine, bromine,iodine) are capable of relatively easily substituting for hydrogenatoms, and as such will bond with the carbons of the core molecule, theyshould result in relatively stable dendrimers. Furthermore, becausefluorine, phosphorous and iodine are monoisotopic, their incorporationinto the dendrimers should further result in a desirable relativelynarrow mass spectral peak.

Additionally, it is preferable to incorporate an element with a largernegative mass defect into the core molecule as it results in a moresubstantial shift in the peak of the mass spectra. The mass defects fora sampling of various elements are provided in TABLE 2.

TABLE 2 Atomic Mass % Isotopic Mass defect Element Isotope Mass (u)Defect comp. per 1000 u Hydrogen ¹H 1.00783 0.00783 99.9885 7.7692 ²H2.01410 0.01410 0.0115 7.0065 Carbon ¹²C 12.00000 0.00000 98.93 0.0000¹³C 13.00335 0.00335 1.07 0.2576 Nitrogen ¹⁴N 14.00307 0.00307 99.6320.2192 ¹⁵N 15.00011 0.00011 0.368 0.0073 Oxygen ¹⁶O 15.99491 −0.0050999.757 −0.3182 ¹⁷O 16.99913 −0.00087 0.038 −0.0512 ¹⁸O 17.99916 −0.000840.205 −0.4667 Fluorine ¹⁹F 18.99840 −0.00160 100 −0.0842 Phosphorus ³¹P30.97377 −0.02623 100 −0.8468 Sulfur ³²S 31.97207 −0.02793 94.93 −0.8736³³S 32.97146 −0.02854 0.76 −0.8656 ³⁴S 33.96787 −0.03213 4.29 −0.9459Chlorine ³⁵Cl 34.96885 −0.03115 75.78 −0.8908 ³⁷Cl 36.96885 −0.0341924.22 −0.9248 Bromine ⁷⁹Br 78.91834 −0.08166 50.69 −1.0347 ⁸¹Br 80.90585−0.08371 49.31 −1.0347 Iodine ¹²⁷I 126.93032 −0.09553 100 −0.7526

Knowing the mass defect for each element, one skilled in the art cancreate a graphical representation of the total population of allpossible peptides (composed of the 20 standard amino acids) per 0.01 uof mass defect for each nominal molecular weight. Such a graphicalrepresentation is shown, in various views, in FIGS. 20-23.

As can be seen in FIGS. 20-23, the graphical representation is definedby two prominent features: (1) an “averagine ridge” and (2) a “scarcinevalley.” The averagine ridge follows the trend of the average amino acid(C_(4.9384)H_(7.7583)N_(1.3577)O_(1.4773)S_(0.0417)) with exactmolecular weight of 111.05431. The scarcine valley represents the leastlikely mass defects for a given molecular weight.

Thus, in use as a calibrant, it is desirable to have a dendrimer tunedso that it has a mass defect that falls within the scarcine valley. Inone embodiment, this can be accomplished by using a dendrimer calibrantwith 2,4,6-triiodalphenol as the core molecule, as shown in Formula 9.

In alternative embodiments, various other tuned core molecules can beutilized. Such core molecules include, but are not limited to,hydroxyl-functional cores such as pentose sugars (linear and furanoseforms), hexaose sugars (linear, pyranose and furanose forms), oligomersof pentose sugars, oligomers of hexose sugars, and cyclodextrins.

Other core molecules may include, but are not limited to,amino-functional cores such as diethylenetriamine[N¹-(2-aminoethyl)ethane-1,2-diamine],N,N′-Bis(3-aminopropyl)ethylenediamine[N¹,N¹′-(ethane 1,2diyl)bis(ethane 1,2 diamine)], bis(hexamethylene)triamine[N¹-(6-aminohexyl)hexane-1,6-diamine], spermidine[N¹-(3-aminopropyl)butane-1,4-diamine], tetraethylenepentamine[N¹,N¹′-(ethane-1,2-diyl)bis(N²-(2-aminoethyl)ethane-1,2-diamine)],spermine [N¹,N¹′-(butane-1,4-diyl)bis(propane-1,3-diamine)],N,N′-bis(2-aminoethyl) 1,3-propanediamine[N¹,N¹′-(propane-1,3-diyl)bis(ethane-1,2-diamine)], andpentaethylenehexamine[N¹-(2-aminoethyl)-N²-(2-((2-((2-((2-aminoethyl)amino)ethyl)amino)ethyl)amino)ethyl) ethane-1,2-diamine].

Yet other core molecules may include, but are not limited to,hydroxyl-functional tertiary amine cores such asN-methyldiethanolamine[2,2′-(methylazadiyl)diethanol],N-ethyldiethanolamine [2,2′-(ethylazadiyl)diethanol],N-propyldiethanolamine [2,2′-(propylazadiyl)diethanol],N-butyldiethanolamine [2,2′-(propylazadiyl)diethanol],N,N-Bis(2-hydroxyethyl)-p-toluidine [2,2′-(p-tolylazadiyl)diethanol],N,N-bis(2-hydroxyethl)-m-toluidine [2,2′-(m-tolylazadiyl)diethanol],N-phenyldiethanolamine [2,2′-(phenylazadiyl) diethanol], triethanolamine[2,2′,2″-nitrilotriethanol], 1 (N,N-bis(2-hydroxyethyl)amino) 2 propanol[2,2′-((2 hydroxypropyl)azanediyl)diethanol], triisopropanolamine[1,1′,1″-nitrilotris(propan-2-ol)], 3-(dimethylamino)-1,2-propanediol,3-(diethylamino)-1,2-propanediol, 3-(dipropylamino)-1,2-propanediol, 3(diisopropylamino) 1,2 propanediol, 2-bis(2hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (also known asbis-tris), N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine[1,1′,1″,1′″-(ethane-1,2-diylbis(azanetriyl))tetrakis(propan-2-ol)],N,N,N′,N′-tetrakis(2-Hydroxyethyl)ethylenediamine, and pentrol[1,1′,1″,1′″-((((2-hydroxypropyl)azanediyl)bis(ethane-2,1-diyl))bis(azanetriyl))tetrakis(propan-2-ol)].

Further core molecules may include, but are not limited to,amino-functional tertiary amine cores such as tris(aminoethyl)amine[N′,N′-bis(2-aminoethyl)ethane-1,2-diamine] andN,N,N′,N′-tetrakis(3-aminopropyl)-1,4-butanediamine[N¹,N¹′-(butane-1,4-diyl)bis(N¹-(3-aminopropyl) propane-1,3-diamine)].

Additional core molecules may include, but are not limited to, hydroxylor amino functional iodocores such as 2,4,6-triiodophenol,2,4,6-triiodophenyl)methanol, 2,4,6-triiodoaniline,(2,4,6-triiodophenyl)methanamine, iohexol[N¹,N³-bis(2,3-dihydroxypropyl)-5-(N-(2,3-dihydroxypropyl)acetamido)-2,4,6-triiodoisophthalamide],and iodixanol[N¹,N¹′-(2-hydroxypropane-1,3-diyl)bis(N³-(2,3-dihydroxypropyl)-5-(N-(2,3-dihydroxypropyl)acetamido)-2,4,6-triiodoisophthalamide)].

Further additional core molecules may include, but are not limited to,hydroxyl-functional tertiary amine iodocores such as2,2′-((2,4,6-triiodophenyl)azanediyl)diethanol,2,2′-((2,4,6-triiodobenzyl)azanediyl)diethanol and3,3′-(((5-((2,3-dihydroxypropyl)(ethyl)amino)-2,4,6-triiodo-1,3-phenylene)bis(methylene))bis(azanediyl))bis(propane-1,2-diol).

Example 22

General Dendronization Procedure for Preparation of CX-([G-n]Ac_(p))_(z)

The procedure of this EXAMPLE is shown schematically as step “i” of FIG.24 (e.g., the syntheses of: dendrimer 1 from hydroxyl-terminated core;of dendrimer 3 from dendrimer 2; etc.). To a round bottom flask wereadded: a known quantity of either hydroxyl-terminated core (e.g.,2,4,6-triiodolphenol) or of dendrimer (e.g., one having the generalformula CX-([G-(n−1)]OH_(r))_(z), where “r” has a value of 2^((n-1)), asappropriate; 1.1 equivalents (per —OH of hydroxyl-terminated core or ofdendrimer) of the acetonide protected bis-MPA anhydride monomer(2,2,5-trimethyl-1,3-dioxane-5-carboxylic) acid anhydride monomer); and0.1 molar equivalents (per —OH of hydroxyl-terminated core or ofdendrimer) of 4-dimethylaminopyridine (DMAP). The reaction mixture wasdissolved in the minimum amount of pyridine, diluted in twice thatamount (relative to pyridine) of dichloromethane, and the reactionmixture was then stirred vigorously for 4 hours at standard temperatureand pressure. The reaction was monitored periodically by MALDI-TOF MS todetermine the degree of coupling. After complete esterification wasobserved by MALDI-TOF MS, the flask contents were transferred to aseparatory funnel, diluted with dichloromethane, extracted three timeswith 1M aqueous NaHSO₄ (sodium bis sulfate) and three extractions with1M aqueous NaHCO₃ (sodium bicarbonate). The organic layers were reducedin vacuo to concentrate the sample, precipitated into hexanes, andfiltered to yield the benzylidene protected dendrimers,CX-([G-n]Ac_(p))_(z), as a white powdery precipitate. The resultingprecipitate may then be prepared for spectrometric analysis via standardprotocols.

Example 23

General Deprotection Procedure for Preparation of CX-([G-n]OH_(η))_(z)

The procedure of this EXAMPLE is shown schematically as step “ii” ofFIG. 1 (e.g., the syntheses of: dendrimer 2 from dendrimer 1; ofdendrimer 4 from dendrimer 3; etc.). To a round bottom flask, a measuredquantity of CX-([G-n]Ac_(r))_(z), where “r” has a value of 2^((n-1)) wasadded and dissolved in 1:1 toluene/methanol. Dowex® solid phase acidresin was added to this solution and the temperature was adjusted to 70°C. The reaction was stirred vigorously for 2-3 hours, and we speculatethat reduced pressure could be used to expedite the reaction. Thedeprotection was monitored by MALDI-TOF MS and when completed, theDowex®1 was filtered from the reaction using methanol. The filtrate wasthen reduced in vacuo to yield a transparent glassy solid having theformula CX ([G-n]OH_(q))_(z). The resulting filtrate may then beprepared for spectrometric analysis via standard protocols.

Example 24

Synthesis of Iodo-Core Calibrants

The iodo-core dendrimer species of this EXAMPLE 24 are shown in FIG. 24.

Synthesis of C1-([G-1]Ac)₁, dendrimer 1 of FIG. 24: 2,4,6-triiodolphenolwas esterified following the General Dendronization Procedure of EXAMPLE22, using the acetonide-protected Bis-MPA anhydride of EXAMPLE 2 andDMAP to afford C1-([G-1]Ac)₁. Molecular Formula: C₁₄H₁₅I₃O₄. MALDI-TOFMS: Theoretical Exact MW: [M+Na]⁺ m/z=650.80021. Observed MW: [M+Na]⁺m/z=650.66.

Synthesis of C1-([G-1]OH₂)₁, dendrimer 2 of FIG. 24: The acetonideprotected dendrimer 1 was deprotected using DOWEX® solid phase acidresin following the General Deprotection Procedure of EXAMPLE 23, toafford C1-([G-1]OH₂)₁. Molecular Formula: C₁₁H₁₁I₃O₄. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=610.769. Observed MW: [M+Na]⁺m/z=610.753.

Synthesis of C1-([G-2]Ac₂)₁, dendrimer 3 of FIG. 24: The hydroxylateddendrimer 2, was esterified following the General DendronizationProcedure of EXAMPLE 22, using the acetonide-protected Bis-MPA anhydrideof EXAMPLE 2 and DMAP to afford C1-([G-2]Ac₂)₁. Molecular Formula:C₂₇H₃₁I₃O₁₀. MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=922.92620.Observed MW: [M+Na]⁺ m/z=922.97.

Synthesis of C1-([G-2]OH₄)₁, dendrimer 4 of FIG. 24: The acetonideprotected dendrimer 3 was deprotected using DOWEX® solid phase acidresin following the General Deprotection Procedure of EXAMPLE 23, toafford C1-([G-2]OH₄)₁. Molecular Formula: C₂₁H₂₇I₂O₁₀. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=842.864. Observed MW: [M+Na]⁺m/z=842.85.

Synthesis of C1-([G-3]Ac₄)₁, dendrimer 5 of FIG. 24: The hydroxylateddendrimer 4, was esterified following the General DendronizationProcedure of EXAMPLE 22, using the acetonide-protected Bis-MPA anhydrideof EXAMPLE 2 and DMAP to afford C1-([G-3]Ac₄)₁. Molecular Formula:C₅₃H₇₅I₃O₂₂. MALDI-TOF MS: Theoretical Exact MW: [M+Na]⁺ m/z=1467.17817.Observed MW: [M+Na]⁺ m/z=1467.07.

Synthesis of C1-([G-3]OH₈)₁, dendrimer 6 of FIG. 24: The acetonideprotected dendrimer 5 was deprotected using DOWEX® solid phase acidresin following the General Deprotection Procedure of EXAMPLE 23, toafford C1-([G-3]OH₈)₁. Molecular Formula: C₄₁H₅₉I₃O₂₂. MALDI-TOF MS:Theoretical Exact MW: [M+Na]⁺ m/z=1307.050. Observed MW: [M+Na]⁺m/z=1306.98.

The dendrimers (dendrimers 2, 4 and 6) made according to the aboveexamples yield a calibrant that is easily distinguishable from naturallyoccurring peptides and peptidic fragments. “First generation” dendrimer2 (as shown in FIG. 24) has a molecular weight (with sodium counterion)of 610.769 (as demonstrated in FIG. 25). As further demonstrated in FIG.25, the mass spectra peak for the first generation dendrimer fallsclearly to the right of, and does not overlap with, the averaginemolecular weight of 610.277. “Second generation” dendrimer 4 (as shownin FIG. 24) has a molecular weight (with sodium counterion) of 842.864(as demonstrated in FIG. 26). As further demonstrated in FIG. 26, themass spectra peak for the second generation dendrimer falls clearly tothe right of (by approximately 0.5 u), and does not overlap with, theaveragine molecular weight of 842.390. “Third generation” dendrimer 6(as shown in FIG. 24) has a molecular weight (with sodium counterion) of1307.050 (as demonstrated in FIG. 27). As further demonstrated in FIG.27, the mass spectra peak for the third generation dendrimer fallsclearly to the right of, and does not overlap with, the averaginemolecular weight of 1306.618. Additionally, as shown in FIG. 23, whenthe first (labeled as G1), second (labeled as G2) and third generation(labeled as G3) dendrimers are overlayed onto the peptide populationmap, the dendrimers clearly fall within the scarcine ridge.

Example 25

Internal Calibration Test—Endomorphin I

To further verify the utility of the iodo-core calibrants of the presentdisclosure in acquiring accurate MALDI-TOF data with high massresolution, peptide Endomorphin I (H-Try-Pro-Trp-Phe-NH₂, having theformula C₃₄H₃₈N₆O₅ was used in an internal calibration test whereinfirst generation dendrimer was mixed with Endomorphin I(H-Try-Pro-Trp-Phe-NH₂, C₃₄H₃₈N₆O₅). As demonstrated in FIG. 28, thepeak of the first generation iodo-core calibrant is distinct from theEndomorphin I peak.

In another embodiment, the dendrimer calibrants may be tuned to includean amine core. During ionization of the analyte, one counterion isattached per molecule. These counterions include, for example, H, Na,and K. It is desirable, as a calibrant, for the core molecule of thedendrimer to be compatible with various counterions. Because amines havean unbounded pair of electrons, they readily attract a hydrogen ion. Assuch, a core molecule containing an amine will readily attract ahydrogen counterion. Triethanolamine may be used as an amine core, asshown in Formula 10. Alternatives to triethanolamine that are speculatedto also readily attract hydrogen counterions are those shown in Formula11 and Formula 12.

Example 26

Synthesis of Amine-Core Calibrants

The amine-core dendrimer species of this EXAMPLE 26 are shown in FIG.29.

Synthesis of C3-([G-1]Ph)₃, dendrimer 1 of FIG. 29: Triethanolamine, wasesterified following the General Dendronization Procedure of EXAMPLE 3,using the benzylidene-protected Bis-MPA anhydride of EXAMPLE 2 and DMAPto afford C3-([G-1]Ph)₃. Molecular Formula: C₄₂H₅₁NO₁₂. MALDI TOF MS:Theoretical Exact MW: [M+H]⁺ m/z=762.34895. Observed MW: [M+H]⁺m/z=762.04.

Synthesis of C3-([G-1]OH₂)₃, dendrimer 2 of FIG. 29: The benzylideneprotected dendrimer 1 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4 to affordC3-([G-1]OH₂)₃. Molecular Formula: C₂₁H₃₉NO₁₂. MALDI-TOF MS: TheoreticalExact MW: [M+H]⁺ m/z=498.25505. Observed MW: [M+H]⁺ m/z=to bedetermined.

Synthesis of C3-([G-2]Ph₂)₃, dendrimer 3 of FIG. 29: The hydroxylateddendrimer 2, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-2]Ph₂)₃. MolecularFormula: C₉₃H₁₁₁NO₃₀. MALDI-TOF MS: Theoretical Exact MW: [M+H]⁺m/z=1722.72692. Observed MW: [M+H]⁺ m/z=1722.628.

Synthesis of C3-([G-2]OH₄)₃, dendrimer 4 of FIG. 29: The benzylideneprotected dendrimer 3 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 4, to affordC3-([G-2]OH₄)₃. Molecular Formula: C₅₁H₈₇NO₃₀. MALDI-TOF MS: TheoreticalExact MW: [M+H]⁺ m/z=1194.53912. Observed MW: [M+H]⁺ m/z=1194.27.

Synthesis of C3-([G-3]Ph₄)₃, dendrimer 5 of FIG. 29: The hydroxylateddendrimer 4, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-3]Ph₄)₃. MolecularFormula: C₁₉₅H₂₃₁NO₆₆. MALDI-TOF MS: Theoretical Exact MW: [M+H]⁺m/z=3643.48285. Observed MW: [M+H]⁺ m/z=3641.413.

Synthesis of C3-([G-3]OH)₃, dendrimer 6 of FIG. 29: The benzylideneprotected dendrimer 5 was deprotected using 5% Pd(OH)₂/C and hydrogengas following the General Deprotection Procedure of EXAMPLE 23, toafford C3-([G-3]OH). Molecular Formula: C₁₁₁H₁₈₃NO₆₆. MALDI-TOF MS:Theoretical Exact MW: [M+H]⁺ m/z=2587.10724 Observed MW: [M+H]⁺m/z=2587.19.

Synthesis of C3-([G-4]Ph₈)₃, dendrimer 7 of FIG. 29: The hydroxylateddendrimer 6, was esterified following the General DendronizationProcedure of EXAMPLE 3, using the benzylidene-protected Bis-MPAanhydride of EXAMPLE 2 and DMAP to afford C3-([G-4]Ph₈)₃. MolecularFormula: C₁₁₁H₁₈₃NO₆₆. MALDI-TOF MS: Theoretical Exact MW: [M+H]⁺m/z=7484.99471. Observed MW: [M+H]⁺ m/z=7480.

FIG. 30 shows a MALDI-TOF mass spectra of the amine core dendrimer 2 ina α-Cyano-4-hydroxycinnamic acid (CHCA) matrix, and showing the massspectra peaks of the ionized dendrimer. FIG. 31 shows a MALDI-TOF massspectra of the amine core dendrimer 4 in a CHCA matrix, and showing themass spectra peaks of the ionized dendrimer. As can be appreciated fromFIGS. 30 and 31, the second and third generation amine core dendrimercalibrants are readily compatible with various counterions. Thus, thesedendrimers with an amine molecular core result in a more usefulsynthetic dendritic calibrant.

An alternate exemplary amine-core dendrimer species is shown in FIG. 32wherein N,N,N′,N′-Tetrakis(2-hydroxyethyl)ethylenediamine was esterifiedfollowing the General Dendronization Procedure of EXAMPLE 3, using thebenzylidene-protected Bis-MPA anhydride of EXAMPLE 2 and DMAP to affordC4-([G-1]Ph)₄. FIG. 33 shows a MALDI-TOF mass spectra of C4-([G-1]Ph)₄in a α-Cyano-4-hydroxycinnamic acid (CHCA) matrix, and showing the massspectra peaks of the ionized dendrimer. As can be appreciated from FIG.33, this amine core dendrimer calibrants is readily compatible withvarious counterions. Thus, dendrimers with an amine molecular coreresult in a more useful synthetic dendritic calibrant.

Yet another alternate exemplary amine-core dendrimer species is shown inFIG. 34 wherein bis-tris was esterified following the GeneralDendronization Procedure of EXAMPLE 3, using the benzylidene-protectedBis-MPA anhydride of EXAMPLE 2 and DMAP to afford to C5-[G1]-Ph₅. FIG.35 shows a MALDI-TOF mass spectra of C5-([G-1]Ph)₅ in aα-Cyano-4-hydroxycinnamic acid (CHCA) matrix, and showing the massspectra peaks of the ionized dendrimer. As can be appreciated from FIG.35, this amine core dendrimer calibrants is readily compatible withvarious counterions. Thus, dendrimers with an amine molecular coreresult in a more useful synthetic dendritic calibrant.

All references cited in this specification are herein incorporated byreference as though each reference was specifically and individuallyindicated to be incorporated by reference. The citation of any referenceis for its disclosure prior to the filing date and should not beconstrued as an admission that the present disclosure is not entitled toantedate such reference by virtue of prior invention.

It will be understood that each of the elements described above, or twoor more together may also find a useful application in other types ofmethods differing from the type described above. Without furtheranalysis, the foregoing will so fully reveal the gist of the presentdisclosure that others can, by applying current knowledge, readily adaptit for various applications without omitting features that, from thestandpoint of prior art, fairly constitute essential characteristics ofthe generic or specific aspects of this disclosure set forth in theappended claims. The foregoing embodiments are presented by way ofexample only; the scope of the present disclosure is to be limited onlyby the following claims.

What is claimed is:
 1. A method of calibrating a mass spectrometer, themethod comprising: providing a composition comprising a first dendrimercomprising a first core molecule, wherein said first core molecule isselected from the group consisting of: a molecule comprising between 1and 8 alcohol functionalities, a molecule comprising between 1 and 8amine functionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; and a second dendrimercomprising a second core molecule, wherein said second core molecule isselected from the group consisting of: a molecule comprising between 1and 8 alcohol functionalities, a molecule comprising between 1 and 8amine functionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said second coremolecule is at least 2 but no greater than 8; wherein said first coremolecule has a different number of total alcohol functionalities andamine functionalities than said second core molecule; wherein the firstdendrimer and the second dendrimer each have a mass defect fallingwithin a scarcine valley of an average amino acid; wherein the firstcore molecule and the second core molecule each comprise a mass defectmarker selected from the group consisting of phosphorous, iodine,bromine, and combinations thereof; and wherein said composition hasphysical properties; ionizing at least a portion of said composition;collecting data from said ionized portion of said composition; andrelating said data to said physical properties.
 2. A method ofcalibrating a mass spectrometer, the method comprising: providing acomposition comprising a first dendrimer comprising a first coremolecule, wherein said first core molecule is selected from the groupconsisting of: a molecule comprising between 1 and 8 alcoholfunctionalities, a molecule comprising between 1 and 8 aminefunctionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said first coremolecule is at least 2 but no greater than 8; and a second dendrimercomprising a second core molecule, wherein said second core molecule isselected from the group consisting of: a molecule comprising between 1and 8 alcohol functionalities, a molecule comprising between 1 and 8amine functionalities, and a molecule comprising at least one aminefunctionality and at least one alcohol functionality wherein thecombined number of amine and alcohol functionalities of said second coremolecule is at least 2 but no greater than 8; wherein said first coremolecule has a different number of total alcohol functionalities andamine functionalities than said second core molecule; wherein said firstcore molecule comprises three iodine atoms; wherein at least one ofproperties (A) and (B) are satisfied: (A) the first dendrimer and thesecond dendrimer each have a mass defect falling within a scarcinevalley of an average amino acid, and (B) at least one of the first coremolecule and the second core molecule comprises a tertiary aminefunctionality; and wherein said composition has physical properties;ionizing at least a portion of said composition; collecting data fromsaid ionized portion of said composition; and relating said data to saidphysical properties.
 3. The method of calibrating of claim 1 whereinsaid first core molecule comprises 2,4,6-triiodolphenol.
 4. The methodof calibrating of claim 1 wherein at least one of the first coremolecule and the second core molecule comprises a tertiary aminefunctionality.
 5. The method of calibrating of claim 1 wherein the massdefect marker for the first core molecule and for the second coremolecule is iodine.
 6. The method of calibrating of claim 1 wherein thefirst dendrimer and the second dendrimer each have a mass defectdistinct from an averagine ridge of an average amino acid.
 7. The methodof calibrating of claim 6 wherein the average amino acid has an averagechemical formula of C_(4.9384)H_(7.7583)N_(1.3577)O_(1.4773)S_(0.0417)with a molecular weight of 111.05431.
 8. The method of calibrating ofclaim 6 wherein the first dendrimer and the second dendrimer each have amass difference of about 0.2 amu about 0.5 amu relative to the averagineridge.
 9. The method of calibrating of claim 6 wherein the firstdendrimer and the second dendrimer each have a mass difference of about0.3 amu to about 0.5 amu relative to the averagine ridge.
 10. The methodof calibrating of claim 6 wherein the first dendrimer and the seconddendrimer each have a mass difference of about 0.4 amu to about 0.5 amurelative to the averagine ridge.
 11. The method of calibrating of claim6 wherein the first dendrimer and the second dendrimer each have a massdifference of about 0.5 amu relative to the averagine ridge.